USPTO Patents Application 10551504

O P I c

Office de la propriete
intellectuelle du canada

C I P o

Canadian Intellectual
Property Office

(12)(19)(ca) Demande-Application

(21) (Al)

(72) LITTLE, MELVYN, DE
(72)KIPRIYANOV, SERGEJ, DE

(71)DEUTSCHES KREBSFORSCHUNGSZENTRUM STIFTUNGDES

OFFENTLICHEN RECHTS, DE
(5l)Int.Cl. 6 C07K 16/00, C12N 15/63, G01N 33/53, A61K 39/395,

C07K 16/28
(30) 1998/05/05 (198 1 9 8 46.9) DE

(54) CONSTRUCTIONS D'ANTICORPS MULTIVALENTES
(54) MULTIVALENT ANTIBODY CONSTRUCTS

2,331,641

(86) 1999/05/05

(87) 1999/11/11

A

Promoter

Leader

Vh-A V l -B V H -B V l -A

Hls e

Unker 1

Epltop %
EPITOPE "

Linker 3

Linker 2

(57) La presente invention conceme une construction
d'anticorps F v multivalente, comportant au moins quatre

domaines variables qui sont relies l'un a l'autre par
l'intermediaire des segments peptidiques 1, 2 et 3.
L'invention conceme en outre des plasmides
d'expression qui codent pour une telle construction
d'anticorps F ainsi qu'un procede de realisation des

constructions d'anticorps F v et leur utilisation.

(57) The invention relates to a multivalent F y antibody

construct comprising at least four variable domains
which are connected to one another via peptide linkers 1 ,
2 and 3. The invention also relates to expression
plasmids which code for such an F y antibody constmct.

In addition, the invention relates to a method for
producing the F y antibody constructs and to the use

thereof.

\r\r\i ic+rifi C" <5 n i r\ o InHi ic+rw n i ri i

CA 02331641 2000-11-03

PI^T WELTORGANISATION FOR GHSTIGES EIGENTUM

£■ ^ A Internationales BOro

INTERNATIONALE ANMELDUNG VEROFFENTLICHT NACH DEM VERTRAG tFBER DIE
INTERNATIONALE ZUSAMMENARBEIT AUF DEM GEBIET PES PATENTWESENS (PCT)

(51) Internationale Patentklassifikation 6 ;
C07K 16/00

A2

(11) Internationale Veroflentlichungsnummer: WO 99/57150

11. November 1999 (11.11.99)

(43) Internationales

Veroffentlichungsdatum

(21) Internationales Aktenzeichen:

(22) Internationales Anmeldedatum:

PCT/DE99/01350
5. Mai 1999 (05.05.99)

(30) Prioritatsdaten:

198 19 846.9

5. Mai 1998 (05.05.98)

DE

(71) Anmelder (fiir alle Bestimmungsstaaten ausser US):

DEUTSCHES KREBSFORSCHUNGSZENTRUM
STIFTUNG DES GFFENTLICHEN RECHTS [DE/DE];
Im Neuenheimer Feld 280, D-69120 Heidelberg (DE).

(72) Erfinder; und

(75) Erfinder/Anmelder (nur fiir US): LITTLE, Melvyn [GB/DE];
Fritz-von-Briesen-Strasse 10, D-69151 Neekargemund
(DE). KIPRIYANOV. Sergej [RU/DE]; Furtwanglerstrasse
3, D-69121 Heidelberg (DE).

(74) Anwalt: HUBER, Bernard; Huber & SchOssler, Truderinger
Strasse 246, D-81825 MOnchen (DE).

(81) Bestimmungsstaaten: AL, AM, AT, AU, AZ, BA, BB, BG,
BR, BY, CA, CH, CN, CU, CZ, DK, EE, ES, FI, GB, GD,
GE, GH, GM, HR, HU, ID, VL, IN, IS, JP, KE, KG, KP,
KR, KZ. LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW,
ARIPO Patent (GH, GM, KE, LS, MW, SD, SL, SZ, UG,
ZW), eurasisches Patent (AM, AZ, BY, KG, KZ, MD, RU,
TJ, TM), europaisches Patent (AT, BE, CH, CY, DE, DK,
ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI
Patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR,
NE, SN, TD, TG).

Veroffentlicht

Ohne internationalen Recherchenbericht und erneut zu
veroffentlichen nach Erhalt des Berichts.

(54) Title: MULTIVALENT ANTIBODY CONSTRUCTS

(54) Bezeichnung: MULTIVALENTE ANTIKORPER-KONSTRUKTE

(57) Abstract

The invention relates to a multivalent
F v antibody construct comprising at least four
variable domains which are connected to one
another via peptide linkers 1, 2 and 3. The
invention also relates to expression plasmids
which code for such an F v antibody construct.
In addition, the invention relates to a method
for producing the F v antibody constructs and
to the use thereof.

(57) Zusammenfassung

Die vorliegende Erflndung betrifft
ein multivalentes Fv-Antikorper-Konstrukt
mit mindestens vier variablen Domanen,
die tlber die Peptidlinker 1, 2 und 3
miteinander verbunden sind. Ferner betrifft
die Erflndung Expressionsplasmide, die
fur ein solches Fv-Antikorper-Konstrukt
codieren, und ein Verfahren zur Herstellung
der Fy-Antikorper-Konstrukte sowie deren
Verwendung.

Promoter Vh-A V l -B V h -B V l -A His 6

Leader

Linker 1

Epitop 8-
EPITOPE m

Linker 3

Linker 2

Ag

CA 02331641 2000-11-03

Applicant: Deutsches Krebsf orschungszentrum
Attorney's File: K 2675

Multivalent Antibody Constructs

The present invention relates to multivalent F v antibody
constructs, expression plasmids which code for them, and a
method for producing the F v antibody constructs as well as
the use thereof.

Natural antibodies are dimers and are therefore referred to
as bivalent. They have four variable domains, namely two V H
domains and two V L domains. The variable domains serve as
binding sites for an antigen, a binding site being formed
from a V H domain and a V L domain. Natural antibodies
recognize one antigen each, so that they are also referred
to as monospecific. Furthermore, they also have constant
domains which add to the stability of the natural
antibodies. On the other hand, they are also co-responsible
for undesired immune responses which result when natural
antibodies of various animal species are administered
mutually .

In order to avoid such immune responses, antibodies are
constructed which lack the constant domains. In particular,
these are antibodies which only comprise the variable
domains. Such antibodies are designated F v antibody
constructs. They are often available in the form of single-
chain monomers paired with one another.

CA 02331641 2000-11-03

2

However, it showed that F v antibody constructs only have
little stability. Therefore, their usability for therapeutic
purposes is strongly limited.

Thus, it is the object of the present invention to provide
an antibody by means of which undesired immune responses can
be avoided. Furthermore, it shall have a stability which
makes it usable for therapeutic uses.

According to the invention this is achieved by the subject
matters defined in the claims.

Therefore, the subject matter of the present invention
relates to a multivalent F v antibody construct which has
great stability. Such a construct is suitable for diagnostic
and therapeutic purposes.

The present invention is based on the applicant's insights
that the stability of an F v antibody construct can be
increased if it is present in the form of a single-chain
dimer where the four variable domains are linked with one
another via three peptide linkers. The applicant also
recognized that the F v antibody construct folds with itself
when the middle peptide linker has a length of about 10 to
30 amino acids. The applicant also recognized that the F v
antibody construct folds with other F v antibody constructs
when the middle peptide linker has a length of about up to
10 amino acids so as to obtain a multimeric, i.e.
multivalent, F v antibody construct. The applicant also
realized that the F v antibody construct can be multi-
specific .

According to the invention the applicant's insights are
utilized to provide a multi-valent F v antibody construct

CA 02331641 2000-11-03

3

which comprises at least four variable domains which are
linked with one another via peptide linkers 1, 2 and 3.

The expression "F v antibody construct" refers to an antibody
which has variable domains but no constant domains.

The expression "multivalent F v antibody construct" refers to
an F v antibody which has several, but at least four,
variable domains. This is achieved when the single-chain F v
antibody construct folds with itself so as to give four
variable domains, or folds with other single-chain F v
antibody constructs. In the latter case, an F v antibody
construct is given which has 8, 12, 16, etc., variable
domains. It is favorable for the F v antibody construct to
have four or eight variable domains, i.e. it is bivalent or
tetravalent (cf . Fig. 1) . Furthermore, the variable domains
may be equal or differ from one another, so that the
antibody construct recognizes one or several antigens. The
antibody construct preferably recognizes one or two
antigens, i.e. it is monospecific and bispecific,
respectively. Examples of such antigens are proteins CD19
and CD3.

The expression "peptide linkers 1, 3" refers to a peptide
linker adapted to link variable domains of an F v antibody
construct with one another. The peptide linker may contain
any amino acids, the amino acids glycine (G) , serine (S) and
proline (P) being preferred. The peptide linkers 1 and 3 may
be equal or differ from each other. Furthermore, the peptide
linker may have a length of about 0 to 10 amino acids. In
the former case, the peptide linker is only a peptide bond
from the COOH residue of one of the variable domains and the
NH 2 residue of another of the variable domains. The peptide
linker preferably comprises the amino acid sequence GG.

CA 02331641 2000-11-03

4

The expression "peptide linker 2" refers to a peptide linker
adapted to link variable domains of an F v antibody construct
with one another. The peptide linker may contain any amino
acids, the amino acids glycine (G) , serine (S) and proline
(P) being preferred. The peptide linker may also have a
length of about 3 to 10 amino acids, in partiuclar 5 amino
acids, and most particularly the amino acid sequence GGPGS,
which serves for achieving that the single-chain F v antibody
construct folds with other single-chain F v antibody
constructs. The peptide linker can also have a length of
about 11 to 20 amino acids, in particular 15 to 20 amino
acids, and most particularly the amino acid sequence (G 4 S) 4 ,
which serves for achieving that the single-chain F v antibody
construct folds with itself.

An F v antibody construct according to the invention can be
produced by common methods. A method is favorable in which
DNAs coding for the peptide linkers 1, 2 and 3 are ligated
with DNAs coding for the four variable domains of an F v
antibody construct such that the peptide linkers link the
variable domains with one another and the resulting DNA
molecule is expressed in an expression plasmid. Reference is
made to Examples 1 to 6. As to the expressions "F v antibody
construct" and "peptide linker" reference is made to the
above explanations and, by way of supplement, to Maniatis,
T. et al., Molecular Cloning, A Laboratory Manual, Cold
Spring Harbor Laboratory 1982.

DNAs which code for an F v antibody construct according to
the invention also represent a subject matter of the present
invention. Furthermore, expression plasmids which contain
such DNAs also represent a subject matter of the present
invention. Preferred expression plasmids are pDISC3xl9-LL,

CA 02331641 2000-11-03
5

pDISC3xl9-SL, pPIC-DISC-LL, pPIC-DISC-SL, pDISC5-LL and
pDISC6-SL. The first four were deposited with the DSMZ
(Deutsche Sammlung fur Mikroorganismen und Zellen) [German-
type collection for micro-organisms and cells] on April 30,
1998 under DSM 12150, DSM 12149, DSM 12152 and DSM 12151,
respectively.

Another subject matter of the present invention relates to a
kit, comprising:

(a) an F v antibody construct according to the invention,
and/ or

(b) an expression plasmid according to the invention, and

(c) conventional auxiliary agents, such as buffers,
solvents and controls.

One or several representatives of the individual components
may be present.

The present invention provides a multivalent F v antibody
construct where the variable domains are linked with one
another via peptide linkers. Such an antibody construct
distinguishes itself in that it contains no parts which can
lead to undesired immune reactions. Furthermore, it has
great stability. It also enables to bind several antigens
simultaneously. Therefore, the F v antibody construct
according to the invention is perfectly adapted to be used
not only for diagnostic but also for therapeutic purposes.
Such purposes can be seen as regards any disease, in
particular a viral, bacterial or tumoral disease.

CA 02331641 2000-11-03

6

Brief description of the drawings:

Fig. 1 shows the genetic organization of an F v antibody
construct (A) according to the invention and schemes for
forming a bivalent (B) or tetravalent F v antibody construct
(C) . Ag: antigen; His 6 : six C-terminal histidine residues;
stop: stop codon (TAA) ; V H and V L : variable region of the
heavy and light chains.

Fig. 2 shows the scheme for the construction of the plasmids
pDISC3xl9-LL and pDISC3xl9-SL . c-myc: sequence coding for an
epitope which is recognized by the antibody 9E1, His 6 :
sequence which codes for six C-terminal histidine residues;
PelB: signal peptide sequence of the bacterial pectate lyase
(PelB leader); rbs: ribosome binding site; Stop: stop codon
(TAA) ; V H and V L : variable region of the heavy and light
chains .

Fig. 3 shows a diagram of the expression plasmid pDISC3xl9-
LL. 6xHis: sequence which codes for six C-terminal histidine
residues; bla: gene which codes for fi-lactamase responsible
for ampicillin resistance; bp: base pairs; c-myc: sequence
coding for an epitope which is recognized by the 9E10
antibody; ColEl: origin of the DNA replication; fl-IG:
intergenic region of the bacteriophage fl; Lac P/O: wt lac-
operon promoter /operator ; linker 1: sequence which codes for
a GlyGly dipeptide linking the V H and V L domains; linker 2:
sequence coding for a (Gly 4 Ser) 4 polypeptide which links the
hybrid scFv fragments; Pel-B leader: signal peptide sequence
of the bacterial pectate lyase; rbs: ribosome binding site;
V H and V L : variable region of the heavy and light chains.

Fig. 4 shows a diagram of the expression plasmid pDISC3xl9-
SL. 6xHis: sequence which codes for six C-terminal histidine

CA 02331641 2000-11-03
7

residues; bla: gene which codes for B-lactamase which is
responsible for the ampicillin resistance; bp: base pairs;
c-myc: sequence coding for an epitope recognized by the 9E10
antibody; ColEl: origin of DNA replication; fl-IG:
intergenic region of the bacteriophage fl; Lac P/0: wt lac-
operon promoter/operator: linker 1: sequence which codes for
a GlyGly dipeptide which links the V H and V L domains; linker
3: sequence which codes for a GlyGlyProGlySer oligopeptide
which links the hybrid scFv fragments; Pel-B leader: signal
peptide sequence of the bacterial pectate lyase; rbs :
ribosome binding site; V H and V L : variable region of the
heavy and light chains .

Fig. 5 shows the nucleotide sequence and the amino acid
sequence derived therefrom of the bivalent F v antibody
construct encoded by the expression plasmid pDIS3xl9-LL. c-
myc epitope: sequence coding for an epitope which is
recognized by the antibody 9E10; CDR: region determining the
complementarity; framework: framework region; His6 tail:
sequence which codes for six C-terminal histidine residues;
PelB leader: signal peptide sequence of the bacterial
pectate lyase; RBS: ribosome binding site; V H and V L :
variable region of the heavy and light chains.

Fig. 6 shows the nucleotide sequence and the derived amino
acid sequence of the tetravalent F v antibody construct
encoded by the expression plasmid pDISC3xl9-SL . c-myc
epitope: sequence coding for an epitope which is recognized
by the 9E10 antibody; CDR: region determining
complementarity; framework: framework region; His6 tail:
sequence coding for the six C-terminal histidine residues;
PelB leader: signal peptide sequence of the bacterial
pectate lyase; RBS: ribosome binding site; V H and V L :
variable region of the heavy and light chains .

CA 02331641 2000-11-03

8

Fig. 7 shows the nucleotide sequence and the derived amino
acid sequence of a connection between a gene which codes for
an a-factor leader sequence and a gene coding for the
tetravalent F v antibody construct in the Pichia expression
plasmid pPIC-DISC-SL . Alpha-factor signal: leader peptide
sequence of the Saccharomyces cerevisiae-a factor secretion
signal; V H : variable region of the heavy chain*. Rhombs
indicate the signal cleaving sites.

Fig. 8 shows the nucleotide sequence and the derived amino
acid sequence of a connection between a gene coding for an
a-factor leader sequence and a gene which codes for the
bivalent F v antibody construct in the Pichia expression
plasmid pPIC-DISC-LL . Alpha-factor signal: leader peptide
sequence of the Saccharomyces cerevisiae-a factor secretion
signal; V H : variable region of the heavy chain. Rhombs show
the signal cleaving sites.

Fig. 9 shows a diagram of the expression plasmid pDISC5-LL.
6xHis: sequence coding for six C-terminal histidine
residues; bla: gene which codes for (J-lactamase responsible
for ampicillin resistance; bp: base pairs; c-myc: sequence
coding for an epitope which is recognized by the 9E10
antibody; hok-sok: plasmid-stabili zing DNA locus; LacI : gene
which codes for the Lac repressor; Lac P/0: wt lac-operon-
promoter/operator; LacZ': gene which codes for the a-peptide
of B-galactosidase; linker 1: sequence which codes for a
GlyGly dipeptide connecting the V H and V L domains; linker 2:
sequence which codes for a (Gly 4 Ser) 4 polypeptide linking
the hybrid scFv fragments; M13 IG: intergenic region of the
M13 bacteriophage; pBR322ori: origin of DNA replication;
Pel-B leader: signal peptide sequence of the bacterial
pectate lyase; rbs : ribosome binding site which originates

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from the E. coli lacZ gene (lacZ) , from the bacteriophage T7
gene 10 (T7gl0) or from the E. coli skp gene (skp) ; skp:
gene which codes for the bacterial periplasmic factor
Skp/OmpH; tHP: strong transcription terminator; tIPP:
transcription terminator; V H and V L : variable region of the
heavy and light chains.

Fig. 10 shows a diagram of the expression plasmid pDISC6-SL.
6xHis: sequence which codes for six C-terminal histidine
residues; bla: gene which codes for li-lactamase responsible
for ampicillin resistance; bp: base pairs: c-myc : sequence
coding for an epitope which is recognized by the 9E10
antibody; hok-sok: plasmid-stabilized DNA locus; LacI: gene
which codes for the Lac repressor; Lac P/0: wt lac-operon
promoter/operator; LacZ': gene which codes for the a-peptide
of li-galactosidase; linker 1: sequence which codes for a
GlyGly dipeptide which links the V H and V L domains; linker
3: sequence which codes for a GlyGlyProGlySer oligopeptide
linking the hybrid scFv fragments: M13 IG: intergenic region
of the M13 bacteriophage; pBR322ori: origin of DNA
replication; Pel-B leader: signal peptide sequence of the
bacterial pectate lyase; rbs: ribosome binding site
originating from the E. coli lacZ gene (lacZ), from the
bacteriophage T7 gene 10 (T7gl0) or from the E. coli skp
gene (skp) ; skp: gene which codes for the bacterial
periplasmic factor Skp/OmpH; tHP: strong transcription
terminator; tIPP: transcription terminator; V H and V L :
variable region of the heavy and light chains.

The invention is explained by the below examples.

CA 02331641 2000-11-03
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Example 1 : Construction of the plasmids pDISC3xl9-LL and

pDISC3xl9-SL for the expression of bivalent,
bispecific and/or tetravalent, bispecific F v
antibody constructs in bacteria

The plasmids pH0G-aCDl9 and pH0G-dm0KT3 which code for the
scFv fragments derived from the hybridoma HD37 which is
specific to human CD19 (Kipriyanov et al., 1996, J. -Immunol.
Meth. 196 , 51-62) and from the hybridoma 0KT3 which is
specific to human CD3 (Kipriyanov et al., 1997, Protein
Eng. 10, 445-453) , respectively, were used for the
construction of expression plasmids for a single-chain F v
antibody construct. A PCR fragment 1 of the V H domain of
anti-CD19, followed by a segment which codes for a GlyGly
linker, was produced using the primers DPI, 5'-
TCACACA GAATTC -TTAGATCTATTAAAGAGGAGAAATTAACC, and DP2, 5'-
AGCACAC GATATC ACCGCCAAGCTTGGGTGTTGTTTTGGC (cf . Fig. 2) . The
PCR fragment 1 was cleaved by EcoRI and EcoRV and ligated
with the EcoRI/EcoRV-linearized plasmid pH0G-dm0KT3 so as to
produce the vector pH0G19-3. The PCR fragment 2 of the V L
domain of anti-CD19, followed by a segment which codes for a
c-myc epitope and a hexahistidinyl tail, was produced using
the primers DP3, 5 ' -AGCACAC AAGCTT GGCGGTGATATCTTGCTCACCCAAAC-
TCCA, and DP4 , 5 1 -AGCACACTCTAGAGACACAC AGATCT TTAGTGATGGTGAT-
GGTGATGTGAGTTTAGG . The PCR fragment 2 was cleaved by Hindi I I
and Xbal and ligated with the HIndlll/Xbal-linearized
plasmid pH0G-dm0KT3 so as to obtain the vector pHOG3-19 (cf.
Fig. 2) . The gene coding for the hybrid scFv-3-19 in the
plasmid pHOG3-19 was amplified by means of PCR with the
primers Bi3sk, 5 ' -CAGCCGG CCATGG CGCAGGTGCAACTGCAGCAG and
either Li-1, 5 ' -TATATACTG CAGCTG CACCTGGCTACCACCACCACCGGAGCCG-
CCACCACCGCTACCACCGCCGCCAGAACCACCACCACCAGCGGCCGCAGCATCAGCCCG,
for the production of a long flexible (Gly 4 Ser) 4 inter-scFV
linker (PCR fragment 3, cf. Fig. 2) or Li-2, 5 ' -TATATA-

CA 02331641 2000-11-03
11

CTG CAGCTG CACCTGCGACCCTGGGCCACCAGCGGCCGCAGCATCAGCCCG, for the
production of a short rigid GGPGS linker (PCR fragment 4,
cf. Fig. 2). The expression plasmids pDISC3xl9-LL and
pDISC3xl9-SL were constructed by ligating the NcoI/PvuII
restriction fragment from pHOG19-3, comprising the vector
framework and the Ncol /PvuII-cleaved PCR fragments 3 and 4,
respectively (cf. Figs. 3, 4). The complete nucleotide and
protein seguences of the bivalent and tetravalent F v
antibody constructs are indicated in Figs 5 and 6,
respectively.

Example 2 : Construction of the plasmids pPIC-DISC-LL and

pPIC-DISC-SL for the expression of bivalent,
bispecific and/or tetravalent, bispecific F v
antibody constructs in yeast

(A) Construction of pPIC-DISC-SL

The vector pPICZocA (Invitrogen BV, Leek, Netherlands) for
the expression and secretion of recombinant proteins in the
yeast Pichia pastoris was used as a starting material. It
contains a gene which codes for the Saccharomyces cerevisiae
a-factor secretion signal, followed by a polylinker. The
secretion of this vector is based on the dominant selectable
marker, Zeocin™ which is bifunctional in both Pichia and E.
coli. The gene which codes for the tetravalent F v antibody
construct (scDia-SL) was amplified by means of PCR by the
template pDISC3xl9-SL using the primers 5-PIC, 5'-
CCGT GAATTC CAGGTGCAACTGCAGCAGTCTGGGGCTGAACTGGC, and pSEXBn
5 ' -GGTCGACGTTAACCGACAAACAACAGATAAAACG. The resulting PCR
product was cleaved by EcoRl and Xbal and ligated in
EcoRI/Xbal-linearized pPICZaA. The expression plasmid pPIC-
DISC-SL was obtained. The nucleotide and protein seguences

CA 02331641 2000-11-03

12

of the tetravalent F v antibody construct are shown in Fig.
7.

(B) Construction of pPIC-DISC-LL

The construction of pPIC-DISC-LL was carried out on the
basis of pPICZaA (Invitrogen BV, Leek, Netherlands) and
pDISC3xl9-LL (cf. Fig. 3). The plasmid-DNA pPICZaA was
cleaved by EcoRI . The overhanging 5' -ends were filled using
a Klenow fragment of the E. coli DNA polymerase I. The
resulting DNA was cleaved by Xbal, and the large fragment
comprising the pPIC vector was isolated. Analogous thereto
the DNA of pDISC3xl9-LL was cleaved by Ncol and treated with
a Klenow fragment. Following the cleavage using Xbal a small
fragment, comprising a gene coding for the bivalent F v
antibody, was isolated. Its ligation with a pPIC-derived
vector-DNA resulted in the plasmid pPIC-DISC-LL . The
nucleotide and protein sequences of the bivalent F v antibody
construct are shown in Fig. 8.

Example 3 : Expression of the tetravalent and/or bivalent

F v antibody construct in bacteria

E. coli XLl-blue cells (Strategene, La Jolla, CA) which had
been transformed with the expression plasmids pDISC3xl9-LL
and pDISC3xl9-SL, respectively, were cultured overnight in
2xYT medium with 50 ug/ml ampicillin and 100 mM glucose
(2xYT Ga ) at 37 °C. 1:50 dilutions of the overnight cultures
in 2xYT GA were, cultured as flask cultures at 37 °C while
shaking with 200 rpm. When the cultures had reached an OD 60 o
value of 0.8, the bacteria were pelleted by 10-minute
centrif ugation with 1500 g at 20°C and resuspended in the
same volume of a fresh 2xYT medium containing 50 ug/ml
ampicillin and 0.4 M saccharose. IPTG was added up to a

CA 02331641 2000-11-03
13

final concentration of 0.1 mM, and the growth was continued
at room temperature (20-22 °C) for 18 - 20 h. The cells were
harvested by 10-minute centrif ugation with 5000 g at 4°C.
The culture supernatant was held back and stored on ice. In
order to isolate the soluble periplasmic proteins, the
pelleted bacteria were resuspended in 5 % of the initial
volume of ice-cold 50 mM Tris-HCl, 20 % saccharose, 1 mM
EDTA, pH 8.0. Following 1 hour of incubation on -ice with
occasional stirring the spheroplasts were centrifuged with
30,000 g at 4°C for 30 minutes, the soluble periplasmic
extract being obtained as supernatant and the spheroplasts
with the insoluble periplasmic material being obtained as
pellet. The culture supernatant and the soluble periplasmic
extract were combined and clarified by further
centrifugation (30,000 g, 4°C, 40 min.). The recombinant
product was concentrated by ammonium sulfate precipitation
(final concentration 70 % saturation) . The protein
precipitate was obtained by centrifugation (10,000 g, 4°C,
40 min.) and dissolved in 10 % of the initial volume of 50
mM Tris-HCl, 1 M NaCl, pH 7.0. An immobilized metal affinity
chromatography (IMAC) was carried out at 4°C using a 5 ml
column of chelating sepharose (Pharmacia) which was charged
with Cu 2+ and had been eguilibrated with 50 mM Tris-HCl, 1 M
NaCl, pH 7.0 (starting buffer). The sample was loaded by
passing it over the column. It was then washed with twenty
column volumes of starting buffer, followed by starting
buffer with 50 mM imidazole until the absorption at 280 nm
of the effluent was at a minimum (about thirty column
volumes) . The absorbed material was eluted with 50 mM Tris-
HCl, 1 M NaCl, 250 mM imidazole, pH 7.0.

The protein concentrations were determined with the Bradford
dye binding test (1976, Anal. Biochem. 72, 248-254) using
the Bio-Rad (Munich, Germany) protein assay kit. The

and bivalent F v
the A 2 8o values
1.96 and 1.93,

Example 4 : Expression of the tetravalent and/or bivalent

antibody construct in the yeast Plchla.
pas torts

Competent P. pastoris GS155 cells (Invitrogen) were
electroporated in the presence of 10 pg plasmid-DNA of pPIC-
DISC-LL and pPIC-DISC-SL, respectively, which had been
linearized with Sacl. The transf ormants were selected for 3
days at 30°C on YPD plates containing 100 ug/ml Zeocin™.
The clones which secreted the bivalent and/or tetravalent F v
antibody constructs were selected by plate screening using
an anti-c-myc-mAk 9E10 (IC Chemikalien, Ismaning, Germany).

For the expression of the bivalent F v antibody constructs
and tetravalent F v antibody constructs, respectively, the
clones were cultured in YPD medium in shaking flasks for 2
days at 30°C with stirring. The cells were centrifuged
resuspended in the same volume of the medium containing
methanol and incubated for another 3 days at 30°C with
stirring. The supernatants were obtained after the
centrif ugation . The recombinant product was isolated by
ammonium sulfate precipitation, followed by IMAC as
described above.

Example 5: Characterization of the tetravalent F v

antibody construct and bivalent F v antibody
construct, respectively,

CA 02331641 2000-11-03

14

concentrations of the purified tetravalent
antibody constructs were determined from
using the extinction coefficients e lmg/ml =
respectively.

(A) Size exclusion chromatography

CA 02331641 2000-11-03

15

An analytical gel filtration of the F v antibody constructs
was carried out in PBS using a superdex 200-HR10/30 column
(Pharmacia). The sample volume and the flow rate were 200
ul/min and 0.5 ml/min, respectively. The column was
calibrated with high-molecular and low-molecular gel
filtration calibration kits (Pharmacia) .

(B) Flow cytometry

The human CD3 + /CD1 9"-acute T-cell leukemia line Jurkat and
the CD19 + /CD3" B-cell line JOK-1 were used for flow
cytometrie. 5 x 10 5 cells in 50 ul RPMI 1640 medium (GIBCO
BRL, Eggestein, Germany) which was supplemented with 10 %
FCS and 0.1 % sodium azide (referred to as complete medium)
were incubated with 100 ul of the F v antibody preparations
for 45 minutes on ice. After washing using the complete
medium the cells were incubated with 100 ul 10 ug/ml anti-c-
myc-Mak 9E10 (IC Chemikalien) in the same buffer for 45 min
on ice. After a second wash cycle, the cells were incubated
with 100 ul of the FITC-labeled goat-anti-mouse-IgG (GIBCO
BRL) under the same conditions as before. The cells were
then washed again and resuspended in 100 ul 1 ug/ml
propidium iodide solution (Sigma, Deisenhofen, Germany) in
complete medium with the exclusion of dead cells. The
relative fluorescence of the stained cells was measured
using a FACScan flow cytometer (Becton Dickinson, Mountain
View, CA) .

(C) Cytotoxicity test

The CD19-expressing Burkitt lymphoma cell line Raji and
Namalwa were used as target cells. The cells were incubated
in RPMI 1640 (GIBCO BRL) which was supplemented with 10 %

CA 02331641 2000-11-03
16

heat-inactivated FCS (GIBCO BRL) , 2 mM glutamine and 1 mM
pyruvate, at 37 °C in a dampened atmosphere with 7.5 % C0 2 .
The cytotoxic T-cell tests were carried out in RPMI-1640
medium supplemented with 10 % FCS , 10 mM HEPES, 2 mM
glutamine, 1 mM pyruvate and 0.05 mM 2 -ME. The cytotoxic
activity was evaluated using a standard [ 51 Cr] release test;
2 x 10 s target cells were labeled with 200 uCi Na[ 51 Cr]0 4
(Amersham-Buchler, Braunschweig, Germany) and washed 4 times
and then resuspended in medium in a concentration of 2 x
10 5 /ml. The effector cells were adjusted to a concentration
of 5 x 10 6 /ml. Increasing amounts of CTLs in 100 ul were
titrated to 10 4 target cells/well or cavity in 50 ul. 50 ul
antibodies were added to each well. The entire test was
prepared three times and incubated at 37°C for 4 h. 100 ul
of the supernatant were collected and tested for [ 51 Cr]
release in a gamma counter (Cobra Auto Gamma; Canberra
Packard, Dreieich, Germany) . The maximum release was
determined by incubation of the target cells in 10 % SDS,
and the spontaneous release was determined by incubation of
the cells in medium alone. The specific lysis (%) was
calculated as: (experimental release - spontaneous
release) / (maximum release - spontaneous release) x 100.

Example 6: Construction of the plasmids pDISC5-LL and

pDISC5-SL for the expression of bivalent,
bispecific and/or tetravalent, bispecific F v
antibody constructs in bacteria by high cell
density fermentation

Expression vectors were prepared which contained the hok/sok
plasmid-free cell suicide system and a gene which codes for
the Skp/OmpH periplasmic factor for a greater production of
recombinant antibodies. The skp gene was amplified by PCR
using the primers skp-1, 5 1 -CGA ATT CTT AAG ATA AGA AGG AGT

CA 02331641 2000-11-03
17

TTA TTG TGA AAA AGT GGT TAT TAG CTG CAG G and skp-2, 5 ' -CGA
ATT AAG CTT CAT TAT TTA ACC TGT TTC AGT ACG TCG G using the
plasmid pGAH317 (Hoick and Kleppe, 1988, Gene 67, 117-124).
The resulting PCR fragment was cleaved by Aflll and Hindlll
and inserted in the Af 111/HindIII-linearized plasmid pHKK
(Horn et al., 1996, Appl. Microbiol. Biotechnol. 46, 524-
532) so as to obtain the vector pSKK. The genes obtained in
the plasmids pDISC3xl9-LL and pDISC3xl. 9-SL and coding for
the scFv antibody constructs were amplified by means of the
primers fe-1, 5 ' -CGA ATT TCT AGA TAA GAA GGA GAA ATT AAC CAT
GAA ATA CC and fe-2, 5 ' -CGA ATT CTT AAG CTA TTA GTG ATG GTG
ATG GTG ATG TGA G. The Xbal/Af Ill-cleaved PCR fragments were
inserted in pSKK before the skp insert so as to obtain the
expression plasmids pDISCS-LL and pDISC6-SL, respectively,
which contain tri-cistronic operons under the control of the
lac promoter/operator system (cf. figs. 9, 10).

CA 02331641 2000-11-03

SEQUENCE RECORD

(1) GENERAL INDICATIONS:

(i) APPLICANT:

(A) NAME: Deutsches Krebsf orschungszentrum

(B) STREET: Im Neuenheimer Feld 280

(C) TOWN: Heidelberg

(E) COUNTRY: Germany

(F) POSTAL CODE: 69120

(ii) TITLE OF THE INVENTION: Multivalent "Antibody
Constructs

(iii) NUMBER OF SEQUENCES: 17

(iv) COMPUTER-READABLE VERSION:

(A) DATA CARRIER: floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, version
#1.30 (EPA)

(2) INDICATIONS AS TO SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1698 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: genome DNA

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no
(ix) FEATURE:

(A) NAME /KEY : CDS

(B) POSITION: 28 . . 1689
(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) POSITION: 28.. 1689

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAATTCATTA AAGAGGAGAA ATTAACC ATG AAA TAC CTA TTG CCT ACG GCA

Met Lys Tyr Leu Leu Pro Thr Ala
1 ' 5

51

CA 02331641 2000-11-03

GCC GCT GGC TTG CTG CTG CTG GCA GCT CAG CCG GCC ATG GCG CAG GTG
Ala Ala Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met Ala Gin Vai
10 15 20

CAA CTG CAG CAG TCT GGG GCT GAA CTG GCA AGA CCT GGG GCC TCA GTG
Gin Leu Gin Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val
25 30 35 40

AAG ATG TCC TGC AAG GCT TCT GGC TAC ACC TTT ACT AGG TAC ACG ATG
Lys Met Ser Cys Lys Ala Ser Gly Tvr Thr Phe Thr Arg Tyr Thr Met-
45 50 55

CAC TGG GTA AAA CAG AGG CCT GGA CAG GGT CTG GAA TGG ATT GGA TAC
His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly Tyr
60 65 70

ATT AAT CCT AGC CGT GGT TAT ACT AAT TAC AAT CAG AAG TTC AAG GAC
He Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gin Lys Phe Lys Asp
75 80 85

AAG GCC ACA TTG ACT ACA GAC AAA TCC TCC AGC ACA GCC TAC ATG CAA
Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin
90 95 100

CTG AGC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GCA AGA
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tvr Cys Ala Arg
105 110 115 120

TAT TAT GAT GAT CAT TAC AGC CTT GAC TAC TGG GGC CAA GGC ACC ACT
Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gin Gly Thr Thr
125 130 " 135

CTC ACA GTC TCC TCA GCC AAA ACA ACA CCC AAG CTT GGC GGT GAT ATC
Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Lys Leu Gly Gly Asp He
140 145 150

TTG CTC ACC CAA ACT CCA GCT TCT TTG GCT GTG TCT CTA GGG CAG AGG
Leu Leu Thr Gin Thr Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg
155 160 165

GCC ACC ATC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT GAT
Ala Thr He Ser Cys Lys Ala Ser Gin Ser Val Asp Tyr Asp Gly Asp
170 175 180

AGT TAT TTG AAC TGG TAC CAA CAG ATT CCA GGA CAG CCA CCC AAA CTC
Ser Tyr Leu Asn Trp Tyr Gin Gin He Pro Gly Gin Pro Pro Lys Leu
185 190 195 200

CTC ATC TAT GAT GCA TCC AAT CTA GTT TCT GGG ATC CCA CCC AGG TTT
Leu He Tyr Asp Ala Ser Asn Leu Val Ser Gly He Pro Pro Arg Phe
205 210 215

AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC AAC ATC CAT CCT GTG
Ser Gly Ser Gly Ser Gly Thr Asd Phe Thr Leu Asn He His Pro Val
220 225 230

CA 02331641 2000-11-03

GAG AAG GTG GAT GCT GCA ACC TAT CAC TGT CAG CAA AGT ACT GAG GAT
Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gin Gin Ser Thr Glu Asp
235 240 245

CCG TGG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC AAA CGG GCT GAT
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg Ala Asp
250 255 260

GCT GCG GCC GCT GGT GGT GGT GGT TCT GGC GGC GGT GGT AGC GGT GGT
Ala Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly-
265 270 275 280

GGC GGC TCC GGT GGT GGT GGT AGC CAG GTG CAG CTG CAG CAG TCT GGG
Gly Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Gin Gin Ser Gly
285 290 295

GCT GAG CTG GTG AGG CCT GGG TCC TCA GTG AAG ATT TCC TGC AAG GCT
Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys He Ser Cys Lys Ala
300 305 310

TCT GGC TAT GCA TTC AGT AGC TAC TGG ATG AAC TGG GTG AAG CAG AGG
Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gin Arg
315 320 325

CCT GGA CAG GGT CTT GAG TGG ATT GGA CAG ATT TGG CCT GGA GAT GGT
Pro Gly Gin Gly Leu Glu Trp lie Gly Gin He Trp Pro Gly Asp Gly
330 335 340

GAT ACT AAC TAC AAT GGA AAG TTC AAG GGT AAA GCC ACT CTG ACT GCA
Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala
345 350 355 360

GAC GAA TCC TCC AGC ACA GCC TAC ATG CAA CTC AGC AGC CTA GCA TCT
Asd Glu Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Ala Ser
365 370 375

GAG GAC TCT GCG GTC TAT TTC TGT GCA AGA CGG GAG ACT ACG ACG GTA
Glu Asd Ser Ala Val Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr Val
380 385 390

GGC CGT TAT TAC TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC TCA GTC
Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Ser Val
395 400 405

ACC GTC TCC TCA GCC AAA ACA ACA CCC AAG CTT GGC GGT GAT ATC GTG
Thr Val Ser Ser Ala Lys Thr Thr Pro Lys Leu Gly Gly Asp He Val
410 415 420

CTC ACT CAG TCT CCA GCA ATC ATG TCT GCA TCT CCA GGG GAG AAG GTC
Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly Glu Lys Val
425 430 435 440

ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG AAC TGG TAC
Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr
445 45.0 455

CA 02331641 2000-11-03

CAG CAG AAG TCA GGC ACC TCC CCC AAA AGA TGG ATT TAT GAC ACA TCC
Gin Gin Lys Ser Gly Thr Ser Pro Lys Arg Trp lie Tyr Asp Thr Ser
460 465 * ' 470

AAA CTG GCT TCT GGA GTC CCT GCT CAC TTC AGG GGC AGT GGG TCT GGG
Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly
475 480 435

ACC TCT TAC TCT CTC ACA ATC AGC GGC ATG GAG GCT GAA GAT GCT GCC
Thr Ser Tyr Ser Leu Thr He Ser Gly Met Glu Ala Glu Asd Ala Ala.
490 495 500

ACT TAT TAC TGC CAG CAG TGG AGT AGT AAC CCA TTC ACG TTC GGC TCG
Thr Tyr Tyr Cys Gin Gin Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser
505 510 515 520

GGG ACA AAG TTG GAA ATA AAC CGG GCT GAT ACT GCA CCA ACT GGA TCC
Gly Thr Lys Leu Glu He Asn Arg Ala Asp Thr Ala Pro Thr Gly Ser
525 530 535

GAA CAA AAG CTG ATC TCA GAA GAA GAC CTA AAC TCA CAT CAC CAT CAC
Glu Gin Lys Leu He Ser Glu Glu Asp Leu Asn Ser His His His His
540 545 550

CAT CAC TAATCTAGA
His His

(2) INDICATIONS AS TO ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 554 amino acids

(B) KIND: amino acid
(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
15 10 15

Ala Gin Pro Ala Met Ala C-ln Val Gin Leu Gin Gin Ser Gly Ala Glu
20 25 30

Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly
35 40 45

Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gin Arg Pro Gly
50 55 50

Gin Gly Leu Glu Trp He Gly Tyr He Asn Pro Ser Arg Gly Tyr Thr
65 70 75 80

CA 02331641 2000-11-03

Asn Tyr Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
85 90 95

Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp
100 105 110

Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu
115 120 125

Asi3 Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr
130 135 140

Thr Pro Lys Leu Gly Glv Asd lie Leu Leu Thr Gin Thr Pro Ala Ser
145 150 155 160

Leu Ala Val Ser Leu Gly Gin Arg Ala Thr lie Ser Cys Lys Ala Ser
165 170 175

Gin Ser Val Asp Tyr Asd Gly Asp Ser Tyr Leu Asn Trp Tyr Gin Gin
180 185 190

lie Pro Gly Gin Pro Pro Lys Leu Leu lie Tyr Asp Ala Ser Asn Leu
195 200 205

Val Ser Gly lie Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
210 ~ 215 220

Phe Thr Leu Asn lie His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr
225 230 235 240

His Cys Gin Gin Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr
245 250 255

Lys Leu Glu lie Lys Arg Ala Asp Ala Ala Ala Ala Gly Gly Gly Gly
260 265 270

Ser Gly Gly Gly Gly Ser Gly Gly Gly Glv Ser Gly Gly Gly Gly Ser
275 280 285

Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
290 295 300

Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tvr Ala Phe Ser Ser Tyr
305 310 315 320

Trp Met Asn Trp Val Lys Gin Arg Pro Glv Gin Gly Leu Glu Trp lie
325 330 335

Gly Gin lie Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
340 345 350

Lvs Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr
355 " 360 365

CA 02331641 2000-11-03

Met Gin Leu Ser Ser Leu Ala Ser Glu Asd Ser Ala Val Tyr Phe Cys
370 375 380

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tvr Tyr Tyr Ala Met Asd
385 390 395 400

Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr
405 410 415

Pro Lys Leu Gly Gly Asd lie Val Leu Thr Gin Ser Pro Ala lie Met
420 425 430

Ser Ala Ser Pro Gly Glu Lvs Val Thr Met Thr Cys Ser Ala Ser Ser
435 440 445

Ser Val Ser Tvr Met Asn Trp Tyr Gin Gin Lys Ser Gly Thr Ser Pro
450 455 460

Lys Arg Trp lie Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala
465 470 475 480

His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie Ser
485 490 495

Glv Met Glu Ala Glu Asp Ala Ala Thr Tvr Tyr Cys Gin Gin Tro Ser
500 505 510

Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Asn Arg
515 520 " 525

Ala Asp Thr Ala Pro Thr Gly Ser Glu Gin Lys Leu lie Ser Glu Glu
530 535 540

Asp Leu Asn Ser His His His His His His
545 550

INDICATIONS AS TO ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1653 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: genome DNA

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no
(ix) FEATURE:

(A) NAME /KEY: CDS

(B) POSITION: 28.. 1644

CA 02331641 2000-11-03

7

(ix) FEATURE:

(A) NAME /KEY: mat_peptide

(B) POSITION: 28.. 1644

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GAATTCATTA AAGAGGAGAA ATTAACC ATG AAA TAC CTA TTG CCT ACG GCA

Mec Lys Tyr Leu Leu Pro Thr Ala
i 5

GCC GCT GGC TTG CTG CTG CTG GCA GCT CAC- CCG C-CC ATG GCG CAG GTC-
Ala Ala-Gly Leu Leu Leu Leu Ala Ala Gin Pro Ala Met Ala Gin Val
10 15 20

CAA CTG CAG CAG TCT GGG GCT GAA CTG GCA AGA CCT GGC- GCC TCA GTG
C-ln Leu Gin Gin Ser Gly Ala Glu Leu Ala Ara Pro Gly Ala Ser Val
25 30 35 40

AAG ATG TCC TGC AAG GCT 'TCT GGC TAC ACC TTT ACT AGG TAC ACG ATG
Lys Met Ser Cvs Lvs Ala Ser Glv Tvr Thr Phe Thr Arc Tvr Thr Met
45 ' 50 " 55

CAC TGG C-TA AAA CAG AGG CCT GGA CAG GGT CTG GAA TGG ATT GGA TAC
His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trt> He Glv Tyr
60 65 70

ATT AAT CCT AGC CGT GGT TAT ACT AAT TAC AAT CAG AAG TTC AAG GAC
He Asn Pro Ser Arg Gly Tvr Thr Asn Tvr Asn Gin Lys Phe Lvs Asa
75 -"30 35

AAG GCC ACA TTG ACT AC A GAC AAA TCC TCC AGC ACA GCC TAC ATG CAA
Lys Ala Thr Leu Thr Thr Asa Lvs Ser Ser Ser Thr Ala Tvr Met Gin
90 95 100

CTG AGC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GCA AGA
Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tvr Tvr Cvs A.la Arg
105 110 115 " 120

TAT TAT GAT GAT CAT TAC AGC CTT GAC TAC TGG GGC CAA GGC ACC ACT
Tyr Tyr Asp Asn His Tyr Ser Leu Asp Tyr Trp Gly Gin Gly Thr Thr
125 130 " ' 135

CTC ACA GTC TCC TCA GCC AAA ACA ACA CCC AAG CTT GGC GGT GAT ATC
Leu Thr Val Ser Ser Ala Lvs Thr Thr Pro Lys Leu Glv Glv Asd He
140 145 150

TTG CTC ACC CAA ACT CCA GCT TCT TTG GCT GTG TCT CTA GGG CAG AGG
Leu Leu Thr Gin Thr Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg
155 160 155

GCC ACC ATC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT GAT
Ala Thr He Ser Cys Lvs Ala Ser Gin Ser Val Asp Tvr Asd Gly Asp
170 175 130

CA 02331641 2000-11-03

8

AGT TAT TTG AAC TGG TAC CAA CAG ATT CCA GGA CAG CCA CCC AAA CTC

Ser Tyr Leu Asn Trp Tyr Gin Gin He Pro Gly Gin Pro Pro Lys Leu

185 190 195 200

CTC ATC TAT GAT GCA TCC AAT CTA GTT TCT GGG ATC CCA CCC AGG TTT
Leu He Tyr Asp Ala Ser Asn Leu Val Ser Glv He Pro Pro Arg Phe
205 210 " 215

AGT GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC AAC ATC CAT CCT GTG
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn He His Pro Val.
220 225 230

GAG AAG GTG GAT GCT GCA ACC TAT CAC TGT CAG CAA AGT ACT GAG GAT
Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gin Gin Ser Thr Glu Asd
235 240 245

CCG TGG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC AAA CGG GCT GAT
Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys Arg Ala Asp
250 255 260

GCT GCG GCC GCT GGT GGC CCA GGG TCG CAG GTG CAG CTG CAG CAG TCT
Ala Ala Ala Ala Gly Gly Pro Gly Ser Gin Val Gin Leu Gin Gin Ser
265 270 275 280

GGG GCT GAG CTG GTG AGG CCT GGG TCC TCA GTG AAG ATT TCC TGC AAG
Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys He Ser Cys Lys
285 290 " 295

GCT TCT GGC TAT GCA TTC AGT AGC TAC TGG ATG AAC TGG GTG AAG CAG
Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gin
300 305 310

AGG CCT GGA CAG GGT CTT GAG TGG ATT GGA CAG ATT TGG CCT GGA GAT
Arg Pro Gly Gin Gly Leu Glu Trp He Gly Gin He Trp Pro Gly Asp
315 320 325

GGT GAT ACT AAC TAC AAT GGA AAG TTC AAG GGT AAA GCC ACT CTG ACT
Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr
330 335 340

GCA GAC GAA TCC TCC AGC ACA GCC TAC ATG CAA CTC AGC AGC CTA GCA
Ala Asp Glu Ser Ser Ser Thr Ala Tvr Met Gin Leu Ser Ser Leu Ala
345 350 ' 355 360

TCT GAG GAC TCT GCG GTC TAT TTC TGT GCA AGA CGG GAG ACT ACG ACG
Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Glu Thr Thr Thr
365 370 375

GTA GGC CGT TAT TAC TAT GCT ATG GAC TAC TGG GGT CAA GGA ACC TCA
Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp Gly Gin Gly Thr Ser
380 385 390

GTC ACC GTC TCC TCA GCC AAA ACA ACA CCC AAG CTT GGC GGT GAT ATC
Val Thr Val Ser Ser Ala Lys Thr Thr Pro Lys Leu Gly Gly Asp He
395 400 4 0 5'

CA 02331641 2000-11-03

GTG CTC ACT CAG TCT CCA GCA ATC ATG TCT GCA TCT CCA GGG GAG AAG 12 99

Val Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly Glu Lys

410 415 420

GTC ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG AAC TGG 1347

Val Thr Met Thr Cvs Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp

425 430 435 440

TAC CAG CAG AAG TCA GGC ACC TCC CCC AAA AGA TGG ATT TAT GAC ACA 13 95

Tyr Gin Gin Lys Ser Gly Thr Ser Pro Lys Arg Trp He Tyr Asp Ttrr

445 450 455

TCC AAA CTG GCT TCT GGA GTC CCT GCT CAC TTC AC-G GGC AGT GGG TCT 1443

Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser

460 465 470

GGG ACC TCT TAC TCT CTC ACA ATC AGC GGC ATG GAG GCT GAA GAT GCT 1491

Gly Thr Ser Tyr Ser Leu Thr He Ser Gly Met Glu Ala Glu Asp Ala

475 480 485

GCC ACT TAT TAC TGC CAG CAG TGG AGT AGT AAC CCA TTC ACG TTC GGC 1539

Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Ser Asn Pro Phe Thr Phe Gly

490 495 500

TCG GGG ACA AAG TTG GAA ATA AAC CGG GCT GAT ACT GCA CCA ACT GGA 1587

Ser Gly Thr Lys Leu Glu He Asn Arg Ala Asp Thr Ala Pro Thr Gly

505 510 515 520

TCC GAA CAA AAG CTG ATC TCA GAA GAA GAC CTA AAC TCA CAT CAC CAT 163 5

Ser Glu Gin Lvs Leu He Ser Glu Glu Ast> Leu Asn Ser His His His

525 530 535

CAC CAT CAC TAATCTAGA 1653
His His His

(2) INDICATIONS AS TO ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 539 amino acids

(B) KIND: amino acid
(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Lys Tyr Leu
1

Ala Gin Pro Ala

20

Leu Ala Arg Pro
35

Leu Pro Thr Ala

5

Met Ala Gin Val

Gly Ala Ser Val
40

Ala Ala Gly Leu
10

Gin Leu Gin Gin
25

Lys Met Ser Cys

Leu Leu Leu Ala
15

Ser Gly Ala Glu
30

Lys Ala Ser Gly
45

CA 02331641 2000-11-03

10

Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gin Arg Pro Gly
50 55 60

Gin Gly Leu Glu Trp He Gly Tyr He Asn Pro Ser Arg Gly Tyr Thr
65 ' 70 75 80

Asn Tyr Asn Gin Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys
85 90 95

Ser Ser Ser Thr Ala Tyr Met Gin Leu Ser Ser Leu Thr Ser Glu Asp
100 105 HO

Se^ Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu
115 ' 120 125

Asp Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr
130 135 140

Thr Pro Lys Leu Gly Gly Asp He Leu Leu Thr Gin Thr Pro Ala Ser
145 " 150 155 160

Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Lys Ala Ser
165 170 175

Gin Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gin Gin
180 185 190

He Pro Gly Gin Pro Pro Lys Leu Leu He Tyr Asp Ala Ser Asn Leu
195 . 200 205

val Ser Gly He Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

Phe Thr Leu Asn He His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr
225 230 235 240

His Cys Gin Gin Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr
245 250 255

Lys Leu Glu He Lys Arg Ala Asia Ala Ala Ala Ala Gly Gly Pro Gly
260 265 270

Se-r Gin Val Gin Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly
275 280 285

Ser Ser Val Lys He Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser
290 ' 295 300

Tyr Trp Met Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp
305 310 315 320

He Gly Gin He Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys
' 325 330 335

CA 02331641 2000-11-03

11

Phe Lys
Tyr Met

Cys Ala
370

Asp Tyr
385

Thr Pro

Gly Lys Ala
340

Gin Leu Ser
355

Arg Arg Glu
Trp Gly Gin

Thr Leu

Ser Leu

Thr Thr
375

Gly Thr

390

Thr Ala

345

Ala Ser
360

Thr Val

Ser Val

Met Ser

Ser Ser

Pro Lys
450

Ala His
465

Ser Gly
Ser Ser
Arg Ala

Glu Asp
530

Lys Leu Gly
405

Ala Ser Pro
420

Val Ser Tyr
435

Arg Trp lie

Phe Arg Gly

Gly Asp He Val
Gly Glu

Met Glu Ala
485

Asn Pro Phe
500

Asp Thr Ala
515

Leu Asn Ser

Met Asn

Tyr Asp
455

Ser Gly
470

Glu Asp
Thr Phe
Pro Thr

Lvs Val
425

Trp Tyr
440

Thr Ser

Ser Gly
Ala Ala

Gly Ser
505

Gly Ser
520

Asp Glu Ser

Glu Aso Ser

Gly Arg Tyr
380

Thr Val Ser

395

Leu Thr Gin
410

Thr Met Thr

Gin Gin Lys

Lys Leu Ala
460

Thr Ser Tyr

475

Thr Tyr Tyr
490

Gly Thr Lys

Glu Gin Lys

Ser Ser Thr Ala
350

Ala Val Tyr Phe
365

Tyr Tyr Ala Met

Ser Ala Lys Thr
400

Ser Pro Ala He
415

Cys Ser Ala Ser
430

Ser Gly Thr Ser
445

Ser Gly Val Pro

Ser Leu Thr He
480

Cys Gin Gin Trp
495

Leu Glu He Asn
510

Leu He Ser Glu
525

His His
535

His His His His

(2) INDICATIONS AS TO ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

CA 02331641 2000-11-03

12

TATATACTGC AGCTGCACCT GCGACCCTGG GCCACCAGCG GCCGCAGCAT CAGCCCG

(2) INDICATIONS AS TO ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 45 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
CCGTGAATTC CAGGTGCAAC TGCAGCAGTC TGGGGCTGAA CTGGC

(2) INDICATIONS AS TO ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 34 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GGTCGACGTT AACCGACAAA CAACAGATAA AACG

CA 02331641 2000-11-03

13

(2) INDICATIONS AS TO ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 348 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: genome DNA

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no
(ix) FEATURE:

(A) NAME/KEY: CDS

(B) POSITION: 1..348
(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) POSITION: 1..348

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

ATG AGA TTT CCT TCA ATT TTT ACT GCT GTT TTA TTC GCA GCA TCC TCC
Met Arg Phe Pro Ser lie Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15

GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gin
20 25 30

ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA GGG GAT TTC
lie Pro Ala Glu Ala Val lie Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45

GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60

TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA
Phe lie Asn Thr Thr He Ala Ser lie Ala Ala Lys Glu Glu Gly Val
65 70 75 80

TCT CTC GAG AAA AGA GAG GCT GAA GCT GAA TTC CAG GTG CAA CTG CAG
Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Gin Val Gin Leu Gin
85 90 95

CAG TCT GGG GCT GAA CTG GCA AGA CCT GGG GCC TCA GTG AAG ATG TCC
Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser
100 105 110

TGC AAG GCT TCT
Cys Lys Ala Ser
115

CA 02331641 2000-11-03

14

2) INDICATIONS AS TO ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 116 amino acids

(B) KIND: amino acid
(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Arg Phe Pro Ser lie Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gin
20 25 30

lie Pro Ala Glu Ala Val lie Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60

Phe He Asn Thr Thr He Ala Ser He Ala Ala Lys Glu Glu Gly Val
65 70 75 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Gin Val Gin Leu Gin
85 90 95

Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser
100 105 110

Cys Lys Ala Ser
115

(2) INDICATIONS AS TO ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 354 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: genome DNA

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no
(ix) FEATURE:

(A) NAME/KEY: CDS

(B) POSITION: 1..354
(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) POSITION: 1 . . 354

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CA 02331641 2000-11-03

15

ATG AGA TTT CCT TCA ATT TTT ACT GCT GTT TTA TTC GCA GCA TCC TCC
Met Arg Phe Pro Ser He Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15

GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA
Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gin
20 25 " 30

ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA GGG GAT TTC"
He Pro Ala Glu Ala Val He Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 40 45

GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG
Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 50

TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA
Phe He Asn Thr Thr He Ala Ser He Ala Ala Lys Glu Glu Gly Val
55 70 75 80

TCT CTC GAG AAA AGA GAG GCT GAA GCT GAA TTC ATG GCG CAG GTG CAA
Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Met Ala Gin Val Gin
85 90 95

CTG CAG CAG TCT GGG GCT GAA CTG GCA AGA CCT GGG GCC TCA GTG AAG
Leu Gin Gin Ser Gly Ala Glu Leu Ala Arg Pro Glv Ala Ser Val Lys
100 105 110

ATG TCC TGC AAG GCT TCT
Met Ser Cys Lys Ala Ser
115

2) INDICATIONS AS TO ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids

(B) KIND: amino acid
(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Arg Phe Pro Ser He Phe Thr Ala Val Leu Phe Ala Ala Ser Ser
15 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gin
20 25 30

He Pro Ala Glu Ala Val He Gly Tyr Ser Asp Leu Glu Gly Asp Phe
35 - 40 45

CA 02331641 2000-11-03

16

Asd Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu
50 55 60

Phe lie Asn Thr Thr lie Ala Ser lie Ala Ala Lys Glu Glu Gly Val
65 70 75 " 80

Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Met Ala Gin Val Gin
85 90 95

Leu Gin Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys,
100 105 " 110

Met Ser Cvs Lys Ala Ser
115

(2) INDICATIONS AS TO ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

TCACACAC-AA TTCTTAGATC TATTAAAGAG GAGAAATTAA CC

(2) INDICATIONS AS TO ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE- DESCRIPTION: SEQ ID NO: 13:

CA 02331641 2000-11-03

17

AGCACACGAT ATCACCGCCA AGCTTGGGTG TTGTTTTGGC

(2) INDICATIONS AS TO ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14

AGCACACAAG CTTGGCGGTG ATATCTTGCT CACCCAAACT CCA

(2) INDICATIONS AS TO ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15
AGCACACTCT AGAGACACAC AGATCTTTAG TGATGGTGAT GGTGATGTGA GTTTAGG

(2) INDICATIONS AS TO ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

CA 02331641 2000-11-03

18

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

CAGCCGGCCA TGGCGCAGGT GCAACTGCAG CAG 33

(2) INDICATIONS AS TO ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 102 base pairs

(B) KIND: nucleotide

(C) STRAND TYPE: single strand

(D) TOPOLOGY: linear

(ii) KIND OF MOLECULE: other nucleic acid
(A) DESCRIPTION: /desc = "primer"

(iii) HYPOTHETICAL: no

(iv) ANTISENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TATATACTGC AGCTGCACCT GGCTACCACC ACCACCGGAG CCC-CCACCAC CGCTACCACC
GCCGCCAGAA CCACCACCAC CAGCGGCCGC AGCATCAGCC CG

60
102

CA 02331641 2000-11-03

Official File: PCT/DE99/01350
Attorney's File: K 2675

Amended Claims

1. A multivalent F v antibody construct having at least
four variable domains which are linked with one another via
the peptide linkers 1, 2 and 3, wherein the peptide linkers
1 and 3 have 0 to 10 amino acids.

2. The F v antibody construct according to claim 1, wherein
the peptide linkers 1 and 3 have the amino acid sequence GG.

3. The F v antibody construct according to claim 1 or 2,
wherein the F v antibody construct is bivalent.

4. The F v antibody construct according to claim 3, wherein
the peptide linker 2 has 11 to 20 amino acids.

5. The F v antibody construct according to claim 3 or 4,
wherein the peptide linker 2 has the amino acid sequence
(G4S ) 4 .

6. The F v antibody construct according to claim 1 or 2,
wherein the F v antibody construct is tetravalent.

7. The F v antibody construct according to claim 6, wherein
the peptide linker 2 has 3 to 10 amino acids.

CA 02331641 2000-11-03

2

8. The F v antibody construct according to claim 6 or 7,
wherein the peptide linker 2 comprises the amino acid
sequence GGPGS.

9. The F v antibody construct according to any of claims 1
to 8, wherein the F v antibody construct is multispecif ic .

10. F v antibody construct according to claim 9, wherein the
F v antibody construct is bispecific.

11. The F v antibody construct according to any of claims 1
to 8, wherein the F v antibody construct is monospecific.

12. A method of producing the multivalent F v antibody
construct according to any of claims 1 to 11, wherein DNAs
coding for the peptide linkers 1, 2 and 3 are ligated with
DNAS coding for the four variable domains of an F v antibody
construct such that the peptide linkers link the variable
domains with one another and the resulting DNA molecule is
expressed in an expression plasmid.

13. Expression plasmid coding for the multivalent F v
antibody construct according to any of claims 1 to 11.

14. The expression plasmid according to claim 13, namely
pDISC3xl9-LL.

15. The expression plasmid according to claim 13, namely
pDISC3xl9-SL.

16. The expression plasmid according to claim 13, namely
pPIC-DISC-LL.

CA 02331641 2000-11-03

3

17. The expression plasmid according to claim 13, namely
pPIC-DISC-SL.

18. The expression plasmid according to claim 13, namely
pDISC5-LL.

19. The expression plasmid according to claim 13, namely
pDISC6-SL.

20. Use of the multivalent F v antibody construct according
to any of claims 1 to 11 for the diagnosis and/or treatment
of diseases.

21. Use according to claim 20, wherein the diseases are
viral, bacterial or tumoral diseases.

CA 02331641 2000-11-03

5/10

EcoRI RBS PelB

1 GAATTCAT7AAAG2^AGAJLAITA_A^^

I'M K7-_ L ? T A A A G L L L A A Q ?

♦ F-ame-H1 VH anti-CD3

92 CGCAGSTGCAACTGCAGCAGTCTCC^^TTa^
22>A Q V Q L Q Q S G A S L A R ? G A S V K M S C K A S G Y 7 ? "t
C0R-H1 Frame-H2 COR-H2

133 T AGGTACACGATGCAC TGGGTAAAACLAGAGGCCTGGACAGGO^
52> R v T m HWVXQRPGQGLEWIGY I N ? S R G Y ?

Frame-H3

2S~ TAATTACAATCAGAAGTTCAAGGAC >AGr^rACA'^y^*r*f:Ari^^^^'^xrr^r^^r^^riTri-i nCT 1 --' C77-C3~ fT1 C'*.C
30> M 7 N Q K ? K 3 K A T L T T D K S 5 S T A Y M C - 3 S L 7

C0R-H3 F'ame-H4

3 5 4 ATCtGAGCACTCTGC-girTArTACTGT^

109> S £ 3 3 A V V V C A R Y Y D D H Y SID Y W 3 Q G T T L

CH1 Linker 7 Frame-Ll VL anti-CD19

44 0 O.GTC^CTCACC-AW.CA-rACC;A-JGCrr ggcsgtgatatcttgctcacczaa^^

138>T V 5 3 A K 7 T r X L G G D I L 1 T 3 T ? A S L A V 3 L C- Q

COfi-L- F.-ame-L2

530 GGCCriACCLATCTCrTCCAAGGCCAGCCAAAGTGT^^

153 > a A T I 5 C X A S Q = V 3 Y 3 G 3 S Y L M W Y 2 Q I ? G

COR-L2 Frame-L3
514 AGC CAC CCAAACTC CTCATCTA.TG A T G C A T C C A A TCTAGTTTC T GGGATC "ACT" r*--.CA~T"^7 - GTGGC - GTGGG7C7GGG i^CAGA f 7T

1?6>Q ? = :< - z. z ■■: z a s n l v s g : ? = a f s g s g s g t d e

C3R-L3 F-ame-L*
702 C^CCCrTCAACATCCATCrTGTC^L^ G CAAAG T A C T G AGGA r C337GG.-.CGT r ?3GGTGGA

225 > T 1 M I H ? 7 £ X V D A A T Y S C Q Q S T £ D ? W T F G G

Ckacca Notl L/'/t/cer 2

790 GGCACCAAGCTGGAAA.TCAAA CGG3CT3A7G€T GCGG^

2 S 5 > G 7 X L E I X H A D A A A A G G G G S G G G G S G G G G

Pvull rrarne-Hl VH anti-CD19

374 rCCGGTGGTGGTGGTAGCCAGGTC<LV3CTGCV3CAGTCTG^

283 > S G G G G S Q V Q 1 Q Q 3 G A E L V R = G S S V X I S C X

C3R-H1 Frame-H2 CDR-H2

962 CTTC7GGC7ATGCAT7CAGT A GCTACTGGATG AA CT GGGTGAAGCAGAGGC CTGGJ^.CACK3GTCrrTGAGT<^\TTGGA CAGATTTGGC
312 > A 3 G Y A ? S 3 Y W M N W V X Q R ? 3 Q G L £ W : G Q I W

Pstl Frame-H3

104 9 CTGGAGATGGTGATACTAACTACAATGGAAAGTTCAAGGGTA AAr»~^
34i> ? G D G D T M Y M G X ? X G X A 7 L, T A D E 3 3 S T A Y

CDR-H3 '

112 3 TGCAACTCAGC-.GCCTAGCATCTGAC^^
369 > M C ■ L S 3 1 A S I 3 S A V Y ~ C A R R E T T 7 V G R Y Y Y
Frame-Hd CHi Linker 1 Frame-Li

1219 GCTATGGACTACT GGGGTCAAGGAACC*TP.GTCACC3T^
398 > A y. D YWGQGTSVTVSSAXTTPXLG G D I V L T
VL anti-CD3 CDR-L1
1307 AJGTCTCC-jGCAATCAToTCTCCArrC^
427 >q S ? A I M S A 3 ? G E X V 7 M T C 3 A S S 3 V S Y MM W
Frame-L2 CDR-L2 Frame-L3

1393 7accagcacaagtxiac<:<iac:tccc:caaaaga^

456> y q q :< 3 g 7 s ? x r w i y d t 3 x l a s g v ? a r. ? r g

CDR-L3

1481 GTGGGTCTGGGACCTC??TACrXTC^
485>S G SGT S Y 3 L T ~ SGMEAEDAATY YC Q C W S 3 N
Frame-L4 C kappa c-myc epitope

1=63 CCCATTCACG TTCGGCTC 3KjG<3ACAAAGTTGGAAA31^AAC C3GGCTGA7ACTGCA^ GAA CAAAA GC7GATCTCAG

514 > ? ? T ?gsgt:<leintradta?tgs e q X L r s

His5 tail Xbal
163c AAGAAgACCTAAACICA CATCACrATCACCATCACT AATCTS^
343>E E D L N S H H H H H K •

FIGURE 5

CA 02331641 2000-11-03

6/10

EcoRI HBS PelB leader

I GAATTCATTAAAGAGGAGAAATT.AACGA T GAr- i .T. 1 .CCT. 1 .TI

rTGCOITGCTGCTGCTGGC^GCTC.-.G

Ncol
-— -.TGG

? T

1> « X Y L L ? T A A A G L L L L A A Q .= A M
♦ F.-ame-Hl VH anti-CD3

92 CGCAGGTGCAACTGCAGCAGTCTGGG3CI
22> A Q V Q L Q Q S G A E
C0R-H1 Frame-H2
1 3 3 T A GGTACACGATGCAC TGGGT AAAACAGAGGC CTGGACAGGGTCTGGAATGGATTGGLA T A C A T T AA TCCTAGCCGTGGTTATXr
32 > ?. Y T M H W V XQRP32SLEW I C- Y IMPS?. C- Y T

Frame-H3

2£ 7 ^AATTACAATCAGAAGTTCAAGGAC XACC-~ACAT^Ar-y-n^r--A^-^^ir-"^rzr---— irATGeAACTGACOCCCTGAC

30> m y m q :< r :-: ~ x a t i, t t d k = s s t a y m q l s s l t

C0R-H3 F.-ame-ri<:
3 5 4 A.TCTGAGGACTCTGCA-GTCTA^A-CTGTGCAAGA TATTATGATGATCATTACAGCCTTGACTA C TGGGGC3AA.GGCAC dACTC^T -
109 > 3 E D 3 A V Y Y C A ?. y Y D 2 H Y S L D Y W G 0 G 7 T L
CH1 Un/cer J Frame-Li VL anti-CD19

440 CAGTCICCTCAGCGAAAACAACAC"^

123>T V SSAXTTrXlG G DILLTQTPASL A 7 5 1 3 Q

CQF.-L; Frame-12

530 : ^^GC CAC CATCTCCTGC AA GGCCAGCC AAA GTGTTGATTATGA TGGTGATAGTTATTTG AA C TGGTAC CAACAGA.TTC G.AGG AC
1=3 >?. A T I £ C X A £ Q £ 7 D Y 0 G D 5 Y L M W Y Q Q I ? G
_____ C3R-L2 Frame -L3

1?6>Q ? ? X L L Z Y r A 3 N 1 7 £ 31???. rSSSGSGTDF

790 GGCAC3AAGCTGCLAAATCAAAC

CDR-L3

-AGGTGGATGCTOCAACCTATC^C::^! ^
? V E X 7 D A A T Y H C Q Q S T E D ? W T ? G G
Ckacca Motl linker 3 Pvull Frame-Hi

GGCCGCTGG TGGCCCA GGGTC SZAG3TGCAGCTGCAGCAGTCTX3GGGCTGAGCT
255> G ? X L E I K X A D A A A A G G ? G S QVQLQQSGAEL

VH anti-CD19 C0R-H1 Frame-H2

379 C<7TGAGGC:TTC-GG7X:3TCAG7T3A^ G C T A C T G G A T G AA C TGGGTGA-.GCAGAGGC

2S4> V R ? G S S V X I S C X A £ G Y A £ S S Y W M N W V X Q R

CDR-H2

963 CTC<XACAGCX7TCT1T3AGTX£<^TT3<1A CAGATTTGG

314> ? G Q G L E W I G Q I W ? G D G D T N Y N G K ? X G X A
Frame-H3

1051 ACTCTX^C^XAGACGAATCCTCCIVXAC^
342> T 1 T A D E S S 5 T A Y M Q L S S L A S E D S A V Y r C A S
C0R-H3 Frame-FM CH1

1142 GGGAGACTACGACGGTAGGCCGTTATTACTATGCTATGGACTAC TGT^^
372 ► R E T T T V G R Y Y Y A M D Y WGQGTSVTV 33 A K
Linker J F.-ame-Ll VL anti-C03

122 6 CAACACCC AAGCTT G GCGGTXIATATCGTGCTCACTCAGTCTC^
400>T T ? X L G G D Z V L, T 0 S ? A I M 3 A 5 ? G E X V T M T C

C0R-L1 Frame-L2 C0R-L2

13 16 GTGCCAGCTCAAGTGTAAGTTACATGAAC T3GTACCAGCACLAA.GTCAG
430> S A 3 3 3 V S Y M N WYQQXSGTS PKR WIYD T S X
Frame-L3

1401 ACTSQCTTCTGGAGTCCCTTrerir^CTra
4S8> L A S G V P A X E R G S G S G T S Y 3 L. T ISGMEAEDA

CDR-L3 Frame-L4 C kapDa

1491 TGCCACIT&TIftCTGCCAGCAGTGSAGTAOTAA^^
488> A T Y Y C Q Q W S 5 M ? r T E G E G T X L E I N R A D T A
c-myc epitope His6 tail Xbal

1S78 ACCAACT GGATCC GAA CAAAA GCTGATCTCA GAA GAA GA CCTAAACTCA CATCACCATCACG-ATCAC TAATCTAGA
517> ? T G 3 E Q X 1 I £ E E D L N 3 H H H H K H •

FIGURE 6

CA 02331641 2000-11-03

7/10

941 ATGAGATTTCCTTCAATTTTTACTGCTGTTTTATTCGCAGCATC

!► M R F P S I FTAVLFAASSA LAAPVN TT

alpha-factor signal

1015 AAC a .GAAGATGAAACG\3CACAA-.TTCC^

25> TEDETA QI PAEAVI GYSDLEGDFD

1089 TTGCTGTTTTGGCAITTTCCAACAGCACA\A^

50>VAVLPFSNSTNNGLLFI NTTIASIA

EcoRI

Xhol ♦ ♦

1163 GCTXAAGAAGAAGGGGTATCTCTCG^GAA^AG

75> AKEEGVSLEKREAEA EF Q V Q L Q Q S

VH anti-CD3

1234 TGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCT

9S> GA ELA RPGASVKMS-CK AS

FIGURE 7

CA 02331641 2000-11-03

8/10

941 AT GAGATTTCCTTCAATTTTTACTGCTGm

1> M RFPSI FTA VLFAA SSA LA A PVN TT

alpha-factor signal

1015 AACAGAAGATGAAA-CGGCACAAATTCCGGCTGAAGCTCTCATCGGTTACTC

25> TEDETA QI PAEAV1 GYSDLEGDFD

BsrDI

1089 TTGCTGTTTTGCC\TTTTCCAA.CAGCACAAATAACGGGT^

50>V AVLPFSNSTNNGLLF I NTTIASIA*

EcoRI

Xhol ♦ ♦

1163 c<:taaagaa.gaagggg?atctc?cgaga^

75>AKEEGVSLEKREAEAEFMA Q V Q L Q

VH anti-CD3

1235 CAGTCTGGGGCTGAACTQGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCT
99> Q S G A ELARPGASVKMSCKAS

FIGURE 8

CA 02331641 2000-11-03

UNSCANNA3LE ITEM
RECEIVED WITH THIS .APPLICATION
(ITEM ON THE 10TH FLOOR ZONE 5 IN THE FILE PREPARATION SECTION)

DOCUMENT RECU AYEC CE . i - DEMAND t
NE P GUV ANT ETRE BaLAVE
(DOCUMENT AU 10 IEME ETAGE AIRE 5 DANS LA SECTION DE L,
PREPARATION DE5 DOSSIERS'

Promoter V H -A V^-B Vh-B

V L -A His 6

Leader

Epitop &

EPITOPE ®

Linker 1

Linker 3

Linker 2